Methods for Measuring the Acute-1-6

Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine OrganismsFifth EditionOctober 2002U.S. Environmental Protection Agency Office of Water (4303T)1200 Pennsylvania Avenue, NWWashington, DC 20460EPA-821-R-02-012DISCLAIMERThe Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and approved this report for publication. Neither the United States Government nor any of its employees, contractors, or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for any third party’s use of or the results of such use of any information, apparatus, product, or process discussed in this report, or represents that its use by such party would not infringe on privately owned rights.iiCONTENTS Pageiii Figures ....................................................................................vi Tables (vii)1.Introduction (1)2.Types of Tests (2)3.Health and Safety (5)General Precautions (5)Safety Equipment (5)General Laboratory and Field Operation (5)Disease Prevention (6)Safety Manuals (6)Waste Disposal (6)4.Quality Assurance (7)Introduction (7)Facilities, Equipment, and Test Chambers (7)Test Organisms (7)Laboratory Water Used for Culturing and Test Dilution Water (7)Effluent Sampling and Sample Handling (8)Test Conditions (8)Quality of Test Organisms (8)Food Quality (8)Acceptability of Acute Toxicity Test Results (9)Analytical Methods (9)Calibration and Standardization (9)Replication and Test Sensitivity (10)Variability in Toxicity Test Results (10)Demonstrating Acceptable Laboratory Performance (18)Documenting Ongoing Laboratory Performance (18)Reference Toxicants (21)Record Keeping (21)5.Facilities and Equipment (22)General Requirements (22)Cleaning Test Chambers and Laboratory Apparatus (23)Apparatus and Equipment for Culturing and Toxicity Tests (23)Reagents and Consumable Materials (24)Test Organisms (26)6.Test Organisms (27)Test Species (27)Sources of Test Organisms (28)Life Stage (29)Laboratory Culturing (29)Holding and Handling Test Organisms (29)Transportation to the Test Site (29)Test Organism Disposal (30)7.Dilution Water (31)Types of Dilution Water (31)Standard, Synthetic Dilution Water (31)CONTENTS (CONTINUED)Use of Receiving Water as Dilution Water (33)Use of Tap Water as Dilution Water (35)Dilution Water Holding (36)8.Effluent and Receiving Water Sampling and Sample Handling (37)Effluent Sampling (37)Effluent Sample Types (37)Effluent Sampling Recommendations (38)Receiving Water Sampling (39)Effluent and Receiving Water Sample Handling, Preservation, and Shipping (39)Sample Receiving (40)Persistence of Effluent Toxicity During Sample Shipment and Holding (40)9.Acute Toxicity Test Procedures (41)Preparation of Effluent and Receiving Water Samples for Toxicity Tests (41)Preliminary Toxicity Range-finding Tests (43)Multi-Concentration (Definitive) Effluent Toxicity Tests (43)Receiving Water Tests (43)Static Tests (44)Flow-Through Tests (45)Number of Test Organisms (45)Replicate Test Chambers (45)Loading of Test Organisms (46)Illumination (46)Feeding (46)Test Temperature (46)Stress (47)Dissolved Oxygen Concentration (47)Test Duration (50)Acceptability of Test Results (50)Summary of Test Conditions for the Principal Test Organisms (50)10.Test Data (67)Biological Data (67)Chemical and Physical Data (67)11.Acute Toxicity Data Analysis (71)Introduction (71)Determination of the LC50 from Definitive, Multi-Effluent-ConcentrationAcute Toxicity Tests (72)The Graphical Method (72)The Spearman-Karber Method (76)The Trimmed Spearman-Karber Method (78)The Probit Method (81)Determination of No-Observed-Adverse-Effect Concentration (NOAEC)from Multi-Concentration Tests, and Determination of Pass or Fail (Pass/Fail)for Single-Concentration (Paired) Tests (83)General Procedure (88)Single Concentration Test (95)Multi-Concentration Test (98)12.Report Preparation and Test Review (109)Report Preparation (109)Introduction (109)Plant Operations (109)ivCONTENTS (CONTINUED)Source of Effluent, Receiving Water, and Dilution Water (109)Test Conditions (110)Test Organisms (110)Quality Assurance (110)Results (110)Conclusions and Recommendations (110)Test Review (111)Sampling and Handling (111)Test Acceptability Criteria (111)Test Conditions (111)Statistical Methods (111)Concentration-Response Relationships (112)Reference Toxicant Testing (112)Test Variability (113)Cited References (114)Bibliography (119)Appendices (124)A.Distribution, Life Cycle, Taxonomy, and Culture Methods (125)A.1.Ceriodaphnia dubia (125)A.2.Daphnia (D. magna and D.pulex) (140)A.3. Mysids (Mysidopsis bahia and Holmesimysis costata) (159)A.4.Brine Shrimp (Artemia salina) (178)A.5.Fathead Minnow (Pimephales promelas) (185)A.6.Rainbow Trout, Oncorhynchus mykiss and Brook Trout, Salvelinus fontinalis (201)A.7.Sheepshead minnow (Cyprinodon variegatus) (209)A.8.Silversides: Inland Silverside (Mendia beryllina),Atlantic Silverside (M. menidia), and TidewaterSilverside (M. peninsulae) (224)B.Supplemental List of Acute Toxicity Test Species (238)C.Dilutor Systems (240)D.Plans for Mobile Toxicity Test Laboratory (253)D.1.Tandem-Axle Trailer (253)D.2.Fifth Wheel Trailer (256)E.Check Lists and Information Sheets (257)E.1.Toxicity Test Field Equipment List (257)E.2. Information Check List for On-Site Industrial or Municipal Toxicity Test (259)E.3.Daily Events Log (264)E.4.Dilutor Calibration Form (265)E.5.Daily Dilutor Calibration Check (266)vFIGURESNumber Page1.Control (cusum) charts (20)2.Approximate times required to replace water in test chambers in flow-through tests (47)3.Rawson's nomograph for obtaining oxygen saturation values in freshwater atdifferent temperatures at sea level (49)4.Example of data sheet for effluent toxicity tests (69)5.Check list on back of effluent toxicity data sheet (70)6.Flowchart for determination of the LC50 for multi-effluent-concentration acutetoxicity tests (73)7.Plotted data and fitted line for graphical method, using all-or-nothing data (75)8.Example of input for computer program for Trimmed Spearman-Karber Method (80)9.Example of output from computer program for Trimmed Spearman-Karber Method (82)10.Example of input for computer program for Probit Method (84)11.Example of output for computer program for Probit Method (85)12.Flowchart for analysis of single-effluent-concentration test data (86)13.Flowchart for analysis of multi-effluent-concentration test data (87)14.Plot of mean survival proportion data in Table 29 (99)viTABLES Number Pagevii1.Intra-laboratory precision of LC50s from static acute toxicity tests withaquatic organisms using reference toxicants (11)2.Intra- and inter-laboratory precision of acute toxicity tests withDaphnia magna , using a standard effluent (12)3.Inter-laboratory precision of acute toxicity tests with aquatic organisms,using reference toxicants (13)4.Interlaboratory study of acute toxicity test precision, 1990: Summary ofresponses using KCL as the reference toxicant (14)5.National inter-laboratory study of acute toxicity test precision, 1991:Summary of responses using reference toxicants (15)6.National interlaboratory study of acute toxicity test precision, 2000: Precision ofLC50 point estimates for reference toxicant, effluent, and receiving water sample types (16)7.Preparation of synthetic freshwater using reagent grade chemicals (33)8.Preparation of synthetic freshwater using mineral water (34)9.Preparation of synthetic seawater using reagent grade chemicals (34)10.Percent unionized NH 3 in aqueous ammonia solutions: Temperatures between15-26E C and pH's 6.0-8.9 (42)11.Oxygen solubility (mg/L) in water at equilibrium with air at 760 mm Hg (48)12.Summary of test conditions and test acceptability criteria for Ceriodaphnia dubiaacute toxicity tests with effluents and receiving waters (Test Method 2002.0) (51)13.Summary of test conditions and test acceptability criteria for Daphnia pulex andD. magna acute toxicity tests with effluents and receiving waters (Test Method 2021.0) (53)14.Summary of test conditions and test acceptability criteria for fathead minnow,Pimephales promelas , acute toxicity tests with effluents and receiving waters (Test Method 2000.0)..5515.Summary of test conditions and test acceptability criteria for rainbow trout,Oncorhynchus mykiss , and brook trout, Salvelinus fontinalis , acute toxicity testswith effluents and receiving waters (Test Method 2019.0) (57)16.Summary of test conditions and test acceptability criteria for mysid, Mysidopsisbahia , acute toxicity tests with effluents and receiving waters (Test Method 2007.0) (59)17.Summary of test conditions and test acceptability criteria for sheepshead minnow,Cyprinodon variegatus , acute toxicity tests with effluents and receiving waters (Test Method 2004.0).61TABLES(CONTINUED)Number Page18.Summary of test conditions and test acceptability criteria for silverside, Menidiaberyllina, M. menidia, and M. peninsulae, acute toxicity tests with effluents andreceiving waters (Test Method 2006.0) (63)19.Summary of test conditions and test acceptability criteria for west coast mysid,Holmesimysis costata, acute toxicity tests with effluents and receiving waters (65)20.Mortality data (number of dead organisms) from acute toxicity tests used inexamples of LC50 determinations (20 organisms in the control and all testconcentrations) (76)21.Coefficients for the Shapiro Wilk's test (90)22.Quantiles of the Shapiro Wilk's test statistic (92)23.Critical values for Wilcoxon's rank sum test five percent significance level (95)24.Data from an acute single-concentration toxicity test with Ceriodaphnia (95)25.Example of Shapiro Wilk's test: Centered observations (96)26.Example of Shapiro Wilk's test: Ordered observations (96)27.Example of Shapiro Wilk's test: Table of coefficients and differences (97)28.Example of Wilcoxon's rank sum test: Assigning ranks to the control and 100%effluent concentrations (98)29.Fathead minnow survival data (100)30.Centered observations for Shapiro Wilk's example (100)31.Ordered centered observations for the Shapiro Wilk's example (101)32.Coefficients and differences for the Shapiro Wilk's example (102)33.ANOVA table (103)34.ANOVA table for Dunnett's Procedure example (105)35.Calculated t values (106)36.Dunnett's "t" values (107)viiiSECTION 1INTRODUCTION1.1 This manual describes acute toxicity tests for use in the National Pollutant Discharge Elimination System (NPDES) Permits Program to identify effluents and receiving waters containing toxic materials in acutely toxic concentrations. With the exception of the Holmesimysis costata Acute Test (Table 19), the methods included in this manual are referenced in Table IA, 40 CFR Part 136 regulations and, therefore, constitute approved methods for acute toxicity tests. They are also suitable for determining the toxicity of specific compounds contained in discharges. The tests may be conducted in a central laboratory or on-site, by the regulatory agency or the permittee. The Holmesimysis costata Acute Test (Table 19) is specific to Pacific Coast waters and is not listed at 40 CFR Part 136 for nationwide use. This method has been proposed but not yet approved at 40 CFR Part 136.1.2 The data are used for NPDES permits development and to determine compliance with permit toxicity limits. Data can also be used to predict potential acute and chronic toxicity in the receiving water, based on the LC50 and appropriate dilution, application, and persistence factors. The tests are performed as a part of self-monitoring permit requirements, compliance biomonitoring inspections, toxics sampling inspections, and special investigations. Data from acute toxicity tests performed as part of permit requirements are evaluated during compliance evaluation inspections and performance audit inspections.1.3 Modifications of these tests are also used in toxicity reduction evaluations and toxicity identification evaluations to identify the toxic components of an effluent, to aid in the development and implementation of toxicity reduction plans, and to compare and control the effectiveness of various treatment technologies for a given type of industry, irrespective of the receiving water (USEPA, 1988a; USEPA, 1988b; USEPA, 1989a; USEPA, 1989b; USEPA, 1991a).1.4 This methods manual serves as a companion to the short-term chronic toxicity test methods manuals for freshwater and marine organisms (USEPA, 2002a; USEPA, 2002b), the NPDES compliance inspection manual (USEPA, 1988c), and the manual for evaluation of laboratories performing aquatic toxicity tests (USEPA, 1991b). In 2002, EPA revised previous editions of each of the three methods manuals (USEPA, 1993a; USEPA, 1994a; USEPA, 1994b).1.5 Guidance for the implementation of toxicity tests in the NPDES program is provided in the Technical Support Document for Water Quality-based Toxics Control (USEPA, 1991c).1.6 The use of any test species or test conditions other than those described in Tables 12-18 in this manual and referenced in Table 1A, 40 CFR 136.3, shall be considered a major modification to the method and subject to application and approval of alternate test procedures under 40 CFR 136.4 and 40 CFR 136.5.1.7 These methods are restricted to use by, or under the supervision of, analysts experience in the use or conduct of, and interpretation of data from, aquatic toxicity tests. Each analyst must demonstrate the ability to generate acceptable test results with the methods using the procedures described in this methods manual.1.8 This manual was prepared in the established EMSL-Cincinnati format (USEPA, 1983a).1SECTION 2TYPES OF TESTS2.1 The selection of the test type will depend on the NPDES permit requirements, the objectives of the test, the available resources, the requirements of the test organisms, and effluent characteristics such as fluctuations in effluent toxicity.2.2 Effluent acute toxicity is generally measured using a multi-concentration, or definitive test, consisting of a control and a minimum of five effluent concentrations. The tests are designed to provide dose-response information, expressed as the percent effluent concentration that is lethal to 50% of the test organisms (LC50) within the prescribed period of time (24-96 h), or the highest effluent concentration in which survival is not statistically significantly different from the control.2.3 Use of pass/fail tests consisting of a single effluent concentration (e.g., the receiving water concentration or RWC) and a control is not recommended. If the NPDES permit has a whole effluent toxicity limit for acute toxicity at the RWC, it is prudent to use that permit limit as the midpoint of a series of five effluent concentrations. This will ensure that there is sufficient information on the dose-response relationship. For example, the effluent concentrations utilized in a test may be: (1) 100% effluent, (2) (RWC + 100)/2, (3) RWC, (4) RWC/2, and (5) RWC/4. More specifically, if the RWC = 50%, appropriate effluent concentrations may be 100%, 75%, 50%, 25%, and 12.5%.2.4 Receiving (ambient) water toxicity tests commonly employ two treatments, a control and the undiluted receiving water, but may also consist of a series of receiving water dilutions.2.5 A negative result from an acute toxicity test does not preclude the presence of chronic toxicity. Also, because of the potential temporal variability in the toxicity of effluents, a negative test result with a particular sample does not preclude the possibility that samples collected at some other time might exhibit acute (or chronic) toxicity.2.6 The frequency with which acute toxicity tests are conducted under a given NPDES permit is determined by the regulatory agency on the basis of factors such as the variability and degree of toxicity of the waste, production schedules, and process changes.2.7 Tests may be static (static non-renewal or static renewal), or flow-through.2.7.1 STATIC TESTS2.7.1.1 Static non-renewal tests - The test organisms are exposed to the same test solution for the duration of the test.2.7.1.2 Static-renewal tests - The test organisms are exposed to a fresh solution of the same concentration of sample every 24 h or other prescribed interval, either by transferring the test organisms from one test chamber to another, or by replacing all or a portion of solution in the test chambers.2.7.2 FLOW-THROUGH TESTS2.7.2.1 Two types of flow-through tests are in common use: (1) sample is pumped continuously from the sampling point directly to the dilutor system; and (2) grab or composite samples are collected periodically, placed in a tank adjacent to the test laboratory, and pumped continuously from the tank to the dilutor system. The flow-through method employing continuous sampling is the preferred method for on-site tests. Because of the large volume (often 400 L/day) of effluent normally required for flow-through tests, it is generally considered too costly and impractical to conduct these tests off-site at a central laboratory.22.8 Advantages and disadvantages of the types of tests are as follows:2.8.1 STATIC NON-RENEWAL TESTS2.8.1.1 Advantages:1.Simple and inexpensive.2.Very cost effective in determining compliance with permit conditions.3.Limited resources (space, manpower, equipment) required; would permit staff to perform manymore tests in the same amount of time.4.Smaller volume of effluent required than for static renewal or flow-through tests.2.8.1.2 Disadvantages:1.Dissolved oxygen (DO) depletion may result from high chemical oxygen demand (COD),biological oxygen demand (BOD), or metabolic wastes.2.Possible loss of toxicants through volatilization and/or adsorption to the exposure vessels.3.Generally less sensitive than static renewal or flow-through tests, because the toxic substancesmay degrade or be adsorbed, thereby reducing the apparent toxicity. Also, there is less chance ofdetecting slugs of toxic wastes, or other temporal variations in waste properties.2.8.2 STATIC-RENEWAL, ACUTE TOXICITY TESTS2.8.2.1 Advantages:1.Reduced possibility of dissolved oxygen (DO) depletion from high chemical oxygen demand(COD) and/or biological oxygen demand (BOD), or ill effects from metabolic wastes fromorganisms in the test solutions.2.Reduced possibility of loss of toxicants through volatilization and/or adsorption to the exposurevessels.3.Test organisms that rapidly deplete energy reserves are fed when the test solutions are renewed,and are maintained in a healthier state.2.8.2.2 Disadvantages:1.Require greater volume of effluent that non-renewal tests.2.Generally less sensitive than flow-through tests, because the toxic substances may degrade or beadsorbed, thereby reducing the apparent toxicity. Also, there is less chance of detecting slugs oftoxic wastes, or other temporal variations in waste properties.2.8.3 FLOW-THROUGH TESTS2.8.3.1 Advantages:1.Provide a more representative evaluation of the acute toxicity of the source, especially if sample ispumped continuously directly from the source and its toxicity varies with time.2.DO concentrations are more easily maintained in the test chambers.3. A higher loading factor (biomass) may be used.4.The possibility of loss of toxicant due to volatilization, adsorption, degradation, and uptake isreduced.32.8.3.2 Disadvantages:rge volumes of sample and dilution water are required.2.Test equipment is more complex and expensive, and requires more maintenance and attention.3.More space is required to conduct tests.4.Because of the resources required, it would be very difficult to perform multiple or overlappingsequential tests.4SECTION 3HEALTH AND SAFETY3.1 GENERAL PRECAUTIONS3.1.1 Development and maintenance of an effective health and safety program in the laboratory requires an ongoing commitment by laboratory management, and includes (1) the appointment of a laboratory health and safety officer with the responsibility and authority to develop and maintain a safety program, (2) the preparation of a formal, written, health and safety plan, which is provided to each laboratory staff member, (3) an ongoing training program on laboratory safety, and (4) regularly scheduled, documented, safety inspections.3.1.2 Collection and use of effluents in toxicity tests may involve significant risks to personal safety and health. Personnel collecting effluent samples and conducting toxicity tests should take all safety precautions necessary for the prevention of bodily injury and illness which might result from ingestion or invasion of infectious agents, inhalation or absorption of corrosive or toxic substances through skin contact, and asphyxiation due to lack of oxygen or presence of noxious gases.3.1.3 Prior to sample collection and laboratory work, personnel must determine that all required safety equipment and materials have been obtained and are in good condition.3.1.4 Guidelines for the handling and disposal of hazardous materials must be strictly followed.3.2 SAFETY EQUIPMENT3.2.1 PERSONAL SAFETY GEAR3.2.1.1 Personnel must use safety equipment, as required, such as rubber aprons, laboratory coats, respirators, gloves, safety glasses, hard hats, and safety shoes.3.2.2 LABORATORY SAFETY EQUIPMENT3.2.2.1 Each laboratory (including mobile laboratories) must be provided with safety equipment such as first aid kits, fire extinguishers, fire blankets, emergency showers, and eye fountains.3.2.2.2 Mobile laboratories should be equipped with a telephone to enable personnel to summon help in case of emergency.3.3 GENERAL LABORATORY AND FIELD OPERATIONS3.3.1 Guidance in Material Safety Data Sheets should be followed for reagents and other chemicals purchased from supply houses. Incompatible materials should not be stored together.3.3.2 Work with effluents must be performed in compliance with accepted rules pertaining to the handling of hazardous materials (see Safety Manuals, Subsection 3.5). Personnel collecting samples and performing toxicity tests should not work alone.3.3.3 Because the chemical composition of effluents is usually only poorly known, they must be considered as potential health hazards, and exposure to them should be minimized. Fume and canopy hoods over the test areas must be used whenever necessary.3.3.4 It is advisable to cleanse exposed parts of the body immediately after collecting effluent samples.53.3.5 All containers must be adequately labeled to indicate their contents.3.3.6 Strong acids and volatile organic solvents employed in glassware cleaning must be used in a fume hood or under an exhaust canopy over the work area.3.3.7 Good housekeeping contributes to safety and reliable results.3.3.8 Electrical equipment or extension cords not bearing the approval of Underwriter Laboratories must not be used. Ground-fault interrupters must be installed in all "wet" laboratories where electrical equipment is used.3.3.9 Mobile laboratories must be properly grounded to protect against electrical shock.3.4 DISEASE PREVENTION3.4.1 Personnel handling samples which are known or suspected to contain human wastes should be immunized against hepatitis B, tetanus, typhoid fever, and polio.3.5 SAFETY MANUALS3.5.1 For further guidance on safe practices when collecting effluent samples and conducting toxicity tests, check with the permittee and consult general industrial safety manuals, including USEPA (1986) and Walters and Jameson (1984).3.6 WASTE DISPOSAL3.6.1 Wastes generated during toxicity testing must be properly handled and disposed of in an appropriate manner. Each testing facility will have its own waste disposal requirements based on local, state, and Federal rules and regulations. It is extremely important that these rules and regulations be known, understood, and complied with by all persons responsible for, or otherwise involved in, performing testing activities. Local fire officials should be notified of any potentially hazardous conditions.6SECTION 4QUALITY ASSURANCE4.1 INTRODUCTION4.1.1 Development and maintenance of a toxicity test laboratory quality assurance (QA) program requires an ongoing commitment by laboratory management, and includes the following: (1) appointment of a laboratory quality assurance officer with the responsibility and authority to develop and maintain a QA program;(2) preparation of a quality assurance plan with data quality objectives; (3) preparation of written descriptions of laboratory standard operating procedures (SOP's) for test organism culturing, toxicity testing, instrument calibration, sample chain-of-custody, laboratory sample tracking system, etc.; and (4) provision of adequate, qualified technical staff and suitable space and equipment to assure reliable data.4.1.2 QA practices within an aquatic toxicology laboratory must address all activities that affect the quality of the final effluent toxicity data, such as: (1) effluent sampling and handling; (2) the source and condition of the test organisms; (3) condition and operation of equipment; (4) test conditions; (5) instrument calibration; (6) replication;(7) use of reference toxicants; (8) record keeping; and (9) data evaluation.4.1.3 Quality control practices, on the other hand, consist of the more focused, routine, day-to-day activities carried out within the scope of the overall QA program. For more detailed discussion of quality assurance, and general guidance on good laboratory practices related to toxicity testing, see: FDA, 1978; USEPA, 1975; USEPA, 1979a; USEPA, 1980a; USEPA, 1980b; USEPA, 1991b; DeWoskin, 1984; and Taylor, 1987.4.1.4 Guidance for the evaluation of laboratories performing toxicity tests and laboratory evaluation criteria may be found in USEPA 1991b.4.2 FACILITIES, EQUIPMENT, AND TEST CHAMBERS4.2.1 Separate test organism culturing and toxicity testing areas should be provided to avoid possible loss of cultures due to cross-contamination. Ventilation systems should be designed and operated to prevent recirculation or leakage of air from chemical analysis laboratories or sample storage and preparation areas into organism culturing or toxicity testing areas, and from toxicity test laboratories and sample preparation areas into culture rooms.4.2.2 Laboratory and toxicity test temperature control equipment must be adequate to maintain recommended test water temperatures. Recommended materials must be used in the fabrication of the test equipment which comes in contact with the effluent (see Section 5, Facilities and Equipment).4.3 TEST ORGANISMS4.3.1 The test organisms used in the procedures described in this manual are listed in Section 6, Test Organisms. The organisms should appear healthy, behave normally, feed well, and have low mortality in cultures, during holding, and in test controls. Test organisms should be positively identified to species.4.4 LABORATORY WATER USED FOR CULTURING AND TEST DILUTION WATER4.4.1 The quality of water used for test organism culturing and for dilution water used in toxicity tests is extremely important. Water for these two uses should come from the same source. The dilution water used in effluent toxicity tests will depend in part on the objectives of the study and logistical constraints, as discussed in detail in Section 7, Dilution Water. The dilution water used for internal quality assurance tests with organisms, food, and reference toxicants should be the water routinely used with success in the laboratory. Types of water are discussed in Section7。